Structural insights into the bi-specific cross-over dual variable antibody architecture by cryo-EM

Multi-specific antibodies (msAbs) are being developed as next generation antibody-based therapeutics. Knowledge of the three-dimensional structures, in the full antibody context, of their fragment antigen-binding (Fab) moieties with or without bound antigens is key to elucidating their therapeutic efficiency and stability. However, the flexibility of msAbs, a feature essential for their multi specificity, has hindered efforts in this direction. Cross-Over Dual Variable immunoglobulin (CODVIg) is a promising bispecific antibody format, designed to simultaneously target the interleukins IL4 and IL13. In this work we present the biophysical and structural characterisation of a CODVFab:IL13 complex in the full antibody context, using cryo-electron microscopy at an overall resolution of 4.2 Å. Unlike the 1:2 stoichiometry previously observed for CODVIg:IL4, CODVIg:IL13 shows a 1:1 stoichiometry. As well as providing details of the IL13-CODV binding interface, including the residues involved in the epitope-paratope region, the structure of CODVFab:IL13 also validates the use of labelling antibody as a new strategy for the single particle cryo-EM study of msAbs in complex with one, or more, antigens. This strategy reduced the inherent flexibility of the IL13 binding domain of CODV without inducing either structural changes at the epitope level or steric hindrance between the IL4 and IL13 binding regions of CODVIg. The work presented here thus also contributes to the development of methodology for the structural study of msAbs, a promising platform for cancer immunotherapy.

Multi-specific antibodies (msAbs) are antibodies engineered to simultaneously and selectively bind to two or more different targets 1,2 . These molecules enable a wide array of therapeutic strategies, such as signalling modulation by soluble or membrane ligand targeting, effector cell retargeting, piggybacking and cofactor mimetization 3 that are not available exploiting conventional monospecific antibodies.
Several bi-specific antibodies (bsAbs) formats have been developed 4 for multitude of biotherapeutic use including cancer 5,6 . Each format has a distinct architecture, molecular range, ligand affinity and half-life in serum, which, in turn, translates into particular advantages and disadvantages for pharmacokinetics, pharmacodynamics, manufacturing, formulation and other drug-design criteria. Indeed, close to 100 bsAbs are currently in development with two already having been brought to market 7 . The formats developed generally fall into two groups: those containing only the relevant fragment antigen-binding (Fab) domains, and those which include a full-antibody construct. Fab fragment-based formats present a greater degree of malleability and adaptability, but have been shown, in many cases, to have suboptimal stability and/or an increased propensity for aggregation, both of which are disadvantageous in drug manufacturing and administration, for which sustained homogeneity and stability are crucial 8 . Increased efforts are therefore being made in the development of full-antibody bsAbs. These make use of antibody constant fragment (Fc) regions as a means of improving stability throughout the drug manufacturing and administration process, while still keeping parental antibody level affinity maturation. The presence of the Fc region also enables the exploration of additional therapeutic strategies such as those involving opsonization and complement-mediated cell immunity 9,10 . www.nature.com/scientificreports/ The main aim of the work described here was to obtain high-resolution details of the various CODV Ig domains, and their relative orientations, in both apo-and antigen bound forms. However, preliminary analysis using both negative stain and cryo-electron microscopy (nsEM (Fig. S1), cryo-EM (Fig. S2) suggested that the inherent flexibility of CODV Ig precluded obtaining high-resolution reconstructions. Moreover, no density for antigens was visible in the nsEM and cryo-EM reconstructions obtained. As the biochemical and biophysical characterisation of CODV Ig in complex with IL4 and/or IL13 clearly suggested high affinity bsAbs:antigen binding (Fig. S2), we labelled the CODV Ig :IL13 complex with a second antibody which recognises a different IL13 epitope than does CODV Ig. This allowed us to obtain a medium resolution (4.2 Å) reconstruction of CODV Fab :IL13 in the full antibody context. While the CODV Ig Fc region is still not visible in the reconstruction obtained due to its intrinsic flexibility, our results provide a structural rationale for the unexpected 1:1 stoichiometry for the CODV Ig :IL13 complex. Our results also suggest that labelling bsAbs:antigen complexes with partner antibodies may provide a general strategy for the cryo-EM analysis of such flexible moieties in the full antibody context.

Results
CODV Ig binding to IL13 exhibits a non-canonical 1:1 stoichiometry. The binding mode and affinity of the interleukins to CODV Ig were characterized a priori to cryo-EM studies by a variety of biophysical methods. For CODV Ig to IL13, results from Size Exclusion Chromatography (SEC) and Mass Photometry (MP) experiments primarily indicate a 1:1 stoichiometry rather than the anticipated 1:2 stoichiometry observed for CODV Ig :IL4 (Fig. S3). Although this is puzzling, this appears to agree with the lower CODV Ig -binding affinity of IL13 as compared to IL4, as determined using Surface Plasmon Resonance (SPR) (Fig. S4 and Table S1). To shed more light on this observation and to understand the in vitro and in vivo discrepancies of the antigens binding 11 , we instigated the structural study of CODV Ig bound to IL13 using cryo-EM. However, a classical methodology, using ns-EM followed by cryo-EM of apo-CODV Ig or CODV Ig bound to either or both interleukins, did not provide reconstructions allowing the elucidation of the structural disposition of the different components. nsEM suffered from a combination of the inherent flexibility of the antibody and the small size of the antigens and uniformly converged to reconstructions with no density for the antigens (Figs. S1 and 1c). Moreover, this flexibility also hindered particle alignment in subsequent cryo-EM refinements (Fig. S2). Nevertheless, our initial EM characterizations allowed us both to characterize the inter-domain flexibility and to develop an image processing workflow that focused solely on the Fab domains.
To obtain structural details of a full antibody-IL3 complex by cryo-EM, we developed a strategy exploiting a partner protein that binds to a different surface of the IL13 antigen than does the CODV Fab . This was achieved using the Fab region of the antibody ReferenceAb (RefAb) 15 . Intriguingly, while SEC and MP analyses didn't provide a strong evidence for 1:2:2 stoichiometry for the CODV Ig :IL13:RefAb Fab ternary complex (TC) (Fig. S5a-c), Small Angle X-Ray Scattering (SAXS) experiments coupled with high-resolution size exclusion chromatography (SEC-SAXS) analysis produced an estimated molecular weight of 220-270 kDa and a maximum distance (D max ) of 307 Å, values consistent with a 1:1:1 stoichiometry (Fig. 1d). This was confirmed by the resulting ab-initio molecular envelope which provides an overall view of the shape of the complex and the disposition of its components (Fig. 1e). More quantitative characterisation of the ternary complex by nsEM revealed that the majority of the particles (89%) clearly exhibited a 1:1:1 stoichiometry for the three components (Fig. S5d,e). Additionally, the low-resolution 3D reconstruction obtained from nsEM ( Fig. S5f) clearly shows, as does the SAXS envelope, IL13 flanked by densities corresponding to CODV Fab and RefAb Fab . These experiments confirmed a reduced flexibility and heterogeneity due to binding of RefAb and prompted a full cryo-EM analysis of the ternary complex (Figs. S5g,h and S6, Table S2).  (Fig. 2b), the CODV Fab :IL13 and IL13:RefAb Fab epitope-paratope regions exhibit the highest resolution while the IL4 binding subdomains of CODV (VH2 and VL2) and both C1 subdomains of CODV and RefAb, located away from the IL13 binding regions, are at more modest resolutions, in the 6-7 Å range (Figs. 2c and S7). Density for the L3 linker region, connecting VH2 to VH1 and for L4 connecting VH1 to CH1, have better defined densities compared to those observed for the L1 and L2 linkers which connect VL1 to VL2 and VL2 to CL, respectively.

Cryo-EM reconstruction of CODV
Although the estimated local resolution of the cryo-EM map at the CODV Fab :IL13 binding interface is better than that of the rest of the molecule (Fig. 2c) it is, nevertheless, still moderate. PDBePISA 16 was thus used to predict atom-atom interactions, the amino acid residues involved in this interface and putative CODV:IL13 H-bonds. These were then confirmed in the cryo-EM density. The reconstruction obtained shows that all 3 CDR loops of CODV VH1 and CDR1 and CDR3 of VL1 are in close proximity to IL13 with higher buried surface area (BSA) compared to rest of the molecule ( Fig. 2d and Table S3). Interestingly, CDR2 of VL1 (Leu-Ala-Ser), exhibits the least buried surface area (BSA) and is not involved in any H-bond interactions with IL13. The heavy chain VH1 shares twice the interface area (564 Å 2 ) with IL13 compared to VL1. Within VH1, CDR3 participates strongly with a maximum BSA of 312Å 2 while CDR1 interacts the least (BSA of 92 Å 2 ). Hydrogen bond interactions follow this trend (Table 1 and Fig. 2e). Here, Asp155 CDR1 and Ser156 CDR1 interact with Ser73 IL13 , Arg180 CDR2 forms hydrogen bonds with Ser76 IL13 and Tyr227 CDR3 hydrogen bonds with Val72 IL13 , Gly74 IL13 and Gln82 IL13 . VL1 makes only three potential hydrogen bonds with IL13, namely Gln34 CDR1 -Arg83 IL13 , Gln34 CDR1 -Ser86 IL13 and Asn95 CDR3 -Arg83 IL13 . All main chain atoms of these interface residues have well-defined EM densities as do all side chains except Gln34 CDR1 , Asn95 CDR3 and Asp155 CDR1 (Fig. 2e). For these latter residues, the side chains have been oriented according to the model refinement criteria. Notably, there are no potential salt-bridges between IL13 and CODV Fab , contrary to what has been observed for IL4 binding to CODV Fab in the crystal structure of www.nature.com/scientificreports/ CODV Fab :IL4 11 . This observation of a lower number of CODV Fab :IL13 interactions compared to CODV Fab :IL4 is commensurate with the lower affinity of CODV Ig for IL13. Importantly though, our reconstruction shows that the CODV Ig IL13 binding site (Fv1) is oriented ~ 90° degrees to that of IL4 (Fv2), with the relevant antigen binding sites more than 50 Å apart (Fig. 3d). This suggests the binding of either of the antigen to CODV Fab is not restricted by the presence or absence of the other antigen. In addition to providing previously unknown details of CODV Ig :IL13 interactions, our reconstruction confirms details of IL13:RefAb Fab paratope-epitope interactions previously observed in the crystal structure of IL13:RefAb Fab 15 . The side chains of most of the residues involved at this interface are in the same orientation as  www.nature.com/scientificreports/ in the crystal structure and fit well in the EM density (Fig. S7). The presence of CODV Ig has thus not perturbed the interaction of IL13 with RefAb Fab .
IL13 binding induces flexibility in the Fab and constant domains of CODV Ig . The relative orientation of variable and constant domains in the Fab allosterically dictates the structural disposition of the Fc domain via the Fab-Fc hinge region. It is thus of critical importance to know the effect of antigen binding on the orientation of these domains as Fc controls the antibody's effector response 9,11 . We therefore investigated if any conformational changes are induced upon RefAb Fab :IL13 binding to CODV Fab , particularly in comparison to the reported crystal structures of apo-CODV Fab 11 and CODV Fab :IL4 11 . www.nature.com/scientificreports/ In the cryo-EM reconstruction of CODV Fab :IL13:RefAb, the lower resolutions observed away from the CODV Fab :IL13 and IL13:RefAb epitope-paratope interfaces are clearly related to the high inter-domain flexibility allowed by the linker regions of the CODV format (Fig. 2c). Principal component analysis 17 suggests rotations and translations of the IL4 binding subdomains of CODV and of the C1 subdomains of CODV and RefAb, while the CODV Fab :IL13 interface, flanked by the two anti-IL13 Fv domains, remains relatively rigid (Fig. 3a-c and Supplementary Video 1). In the movements analysed, the IL4-binding fragment, Fv2 presents a 25° clockwise rotation on the Y axis (Fig. 3a-variability component 1), while the linker, L2, connecting the light chains of C1 and IL4-binding Fv2, seems to allow a small 8 Å outwards translation of C1 resulting in Fv2 moving away from Fv1. In the other major movement observed (Fig. 3b-variability component 2), Fv2 shows an approximately 20° clockwise rotation on the Z axis relative to the IL-13 bound Fv1 and a 45° counter-clockwise rotation around Y axis, further extending the distance and space between the two antigen-binding domains and their binding sites. The observed widening of the IL4 and IL13 paratopes and the intrinsic flexibility and interconnectivity provided by the linkers may aid CODV in rearranging its Fvs, thus facilitating binding of larger ligands, such as HER2 and HER3 located in the cellular membrane, as hypothesised in previous CODV-related work 11 .
In line with the above observation concerning the mobility of CODV Ig C1, a superposition of the cryo-EM reconstruction of CODV Fab :IL13:RefAb Fab with the crystal structures of apo-CODV Fab and CODV Fab :IL4 on Fv1 domain indicates that, upon the binding of IL13:RefAb Fab , the constant domain moves away from both antigen binding sites (Fig. 3d). Upon binding, each antigen Fab seems to push C1 away from the respective variable domains. This movement could be transferred to the Fc domain via the hinge that connects the C1 to the Fc 18,19 . In support of this, in our low resolution cryo-EM reconstructions of the full CODV Ig :IL4, the electron density for Fc is positioned at different orientations relative to the Fab regions (Fig. 3e).

Discussion
Atomic resolution details, including the relative positions of different domains, in particular the variable domains, and their conformations, of the structure of full antibody CODV Ig in complex with IL4 and IL13 will be of valuable information in understanding the CODV Ig framework's capability to simultaneously bind to these two antigens. Existing structural studies include the crystal structures of both apo-CODV Fab and CODV Fab :IL4 11 . However, attempts to crystallize CODV Fab :IL13 or CODV Fab :IL4: IL13 have not yet been successful. We therefore pursued a cryo-EM approach, investigating the structure of CODV Ig :IL13, in a full antibody context, as a precursor to the potential study of CODV Ig :IL4:IL13. The high level of flexibility of the CODV Fc domain means we have only been partially successful, resorting to a combination of labelling and a 'Fab-focused' approaches to produce a cryo-EM reconstruction of a CODV Fab :IL13:RefAb Fab . The results of our structural analysis are intriguing: they clearly suggest that the disposition of the IL4 and IL13 Fab domains should allow the binding of both antigens at the same time to the same Fab. Moreover, our results did not provide a direct suggestion why CODV Ig binds IL13 primarily with a 1:1 stoichiometry rather than the 1:2 ratio observed for CODV Ig :IL4. However, a possible explanation may be found both in our low resolution cryo-EM analysis of CODV Ig :IL4 and CODV Ig :IL4:IL13 complexes (Fig. S2) and in the crystal structures of anti-canine lymphoma Mab231, an IG2a antibody (PDB 1IGT 18 ) and of anti-pheno-barbital (anti-epileptic drug) Mab 61.1.3, an IG1k antibody (PDB 1IGY 19 ). Both in the crystal structures reported and in some of the low resolution classes we observe for CODV Ig :IL4 and CODV Ig :IL4:IL13 complexes (Figs. 3e and S2), the Fc domain is observed in an "inclined" disposition leading to a non-symmetrical interaction of Fc with respect to the Fab fragments. We propose that this propensity of Fc to assume an oblique disposition to one of the Fabs may lead to occlusion of the IL13 binding site in that Fab (Fig. 4). www.nature.com/scientificreports/ Additionally, the crystal structures of full Igs have been proposed to be one of the several structural states that the antibody can assume due to the prevailing internal mobility. As also observed in the structure of Mab231 and elsewhere 20,21 , the Fc domain of CODV Ig also exhibits a substantial degree of internal mobility represented by various classes in 3D classification (Figs. S2 and S6) and due to this fact it was mostly ghosted out in 2D classification. It is not still clear whether this fluctuation in the structural disposition of Fc could be translated to Fab fragments or vice versa with or without antigen binding. Nevertheless, when we compare the domain movements within Fab induced by antigen binding (IL4 or IL13), we observe significant movement of C1 domain relative to the Fv1 and Fv2 domains (Fig. 3d). Whether these antigen-induced movements in the C1 domains of each Fab have a structural influence or place constraints on the disposition of Fc (Fig. 4) requires further investigation. This would require the full antibody structure, perhaps with Fc bound to a third protein and/or with deletion mutants of hinge region. Also, further investigation is necessary to ascertain whether the structural flexibility of Fc and/or the motion observed in the C1 domain can have an effect on the existence of fewer interactions at the epitope-paratope region of CODVFab:IL13 compared to CODVFab:IL4, as fewer protein-protein interactions and subsequently lower affinity could lead to a lower stoichiometry.
In the cryo-EM structure of CODV Fab :IL13:RefAb Fab and in all the cryo-EM reconstructions shown here, the segmental flexibility of Fc in relation to Fab fragments has been routinely observed. It is worth mentioning that the only attempt thus far to obtain structural information using cryo-EM on an intact full bispecific antibody is that for the DVD-Ig IL12XIL18 . Here also, the Fc exhibited huge flexibility such that its density was not observed in many class averages 12 . Although crystal structures exist for intact IgG1 and IgG2 antibodies, they represent a single snapshot from the several structural states the antibodies could adopt 18,19 . Furthermore, several other crystal structures of intact IgG molecules show disordered Fc domains 22 . The only way to restrain this flexibility is to delete the hinge region 23 . Indeed, the length of hinge regions is associated strongly with the available segmental flexibility in an antibody [24][25][26] . However, alterations in hinge length have notable effects on the important effector functions of Fc such as receptor binding, complement activation, etc 23,[26][27][28][29][30] .
Owing to the high affinity of IL4 and IL13 for CODV Ig , we believe that it is unlikely that the ligands separate from the antibody in our experimental conditions and attribute the difficulty in their visualisation by cryo-EM to the small size of the ligand and intrinsic flexibility of the antibody. Attaching RefAb to IL13 seems to limit this flexibility, as evidenced by our 3D classification results. Nevertheless, more experiments, particularly medium-to high-resolution structural studies of the CODV Ig :IL13:IL4 complex, are required to confirm the stoichiometry of IL13 binding to CODV Ig . Despite obtaining stable CODV Ig :IL13:IL4:RefAb by gel filtration, structural studies by EM are currently unsuccessful and the identification of a suitable antibody label for IL4 may be needed.
In conclusion, the work presented here has shown that it is indeed possible to use cryo-EM as a tool for the medium resolution visualisation multi-specific antibody-antigen interfaces within the full-antibody context. However, owing to the intrinsic flexibility of the Fab and Fab-Fc regions, the binding of the small antigens IL4 and IL13 could not be directly imaged either in nsEM or in cryo-EM reconstructions. Indeed, such imaging requires antigen labelling with a molecule of which the binding does not interfere with antibody-antigen binding sites. Here we have used secondary antibody labelling in the cryo-EM study of a multi-specific antibody-antigen complex and this may prove a general approach. Indeed, a similar method has been recently exploited in cryo-EM studies of membrane proteins. Here a synthetic Fab (NabFab) has been used as a label for the small 15 kDa nanobody bound to a membrane protein, thus enhancing the molecular weight to facilitate the cryo-EM analysis 31 . In summary, our cryo-EM reconstruction of CODV Ig :IL13:RefAb Fab ternary complex in the full antibody context confirms that CODV Ig is structurally competent for the simultaneous binding of the interleukins IL4 and IL13 and therefore remains a very promising platform for cancer immunotherapy. The reconstruction appears to confirm a preference, observed in solution, for a 1:1 CODV Ig :IL13 stoichiometry. The study also provides the first visualisation of CODV Ig :IL13 paratope-epitope interactions, providing a structural platform to enhance the therapeutic potential of CODV Ig . Finally, the use of labelling with a secondary antibody to reduce the flexibility of the CODV Fab :IL13 region of the ternary complex and to facilitate FAB-focused particle alignment in cryo-EM analysis suggests this could be used as a general approach for the cryo-EM analysis of full-antibody msAbs. Such an approach has been an important tool for the success of cryo-EM analyses of inherently flexible molecules of high therapeutic interest.

Materials and methods
Preparation and purification of antibodies and antibody-antigen complexes. Cloning procedures for all constructs are explained elsewhere 11 . CODVIg IL4 x IL1 3 (CODV Ig ) was from Human Embrionic Kidney (HEK 293) cells (Invitrogen) and was purified at Sanofi-Aventis Deutschland Gmbh (Frankfurt-am-Main, Germany), as described in 11 . His-tagged IL4 and IL13 proteins were also expressed in HEK 293 cells (Life technologies) and purified at Evotec (France) SAS, by two-step procedure using His-Trap and SEC. Preparations of apo-CODV Ig were stored in His buffer (Histidine 10 mM, NaCl 150 mM, pH 6.0), IL4 and IL13 were on DPBS (5 mM Phosphate, 155 mM NaCl, pH 7.4) and RefAb Fab samples were in TBS (20 mM Tris, 50 mM NaCl, pH 8.0).
For complexes containing only IL4 and/or IL13, these were formed by adding fourfold molar excess of ligand(s) to 1 mg/ml of CODV Ig . CODV Ig :IL4, CODV Ig :IL13 and CODV Ig :IL4:IL13 complexes were purified via size exclusion chromatography (SEC) using a Superdex S200 3.2/300 Increase column equilibrated with SB buffer (50 mM Tris, 95 mM NaCl, 3 mM KCl, pH 8.0) on an ÄKTA Pure (GE Healthcare) at 4 °C. Fractions were collected with an ÄKTA Micro module (GE Healthcare) and those containing CODV Ig :IL4, CODV Ig :IL13 and CODV Ig :IL4:IL13 did not require concentration steps, as they eluted at concentrations in the range of 0.25 to 0.30 mg/ml, suitable for direct grid preparation for cryo-EM. The purified complexes were stored at 4 °C. www.nature.com/scientificreports/ For the purification of the CODV Ig :IL13:RefAb Fab ternary complex (TC), IL13 was first added in fourfold molar excess to CODV Ig at 1 mg/ml and incubated for 15 min. RefAb Fab was then added, also in fourfold molar excess. For purification, a single high-resolution SEC step was performed as outlined above for CODVIg and its complexes with IL14 and/or IL13. As in all the other complexes, only the fractions corresponding to the centre of the peak were collected for cryo-EM grid preparation. The purified ternary complex was stored at 4 °C.
Throughout all purification procedures, eluted fractions were analysed using denaturing 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was checked using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific) in combination with the theoretical absorption coefficients calculated using the PROTPARAM online tool provided by ExPASy 32 .
Characterization of antigen-antibody complexes by mass photometry. All samples were subjected to analysis by mass photometry 33 . Here, 100 nM of CODV Ig , CODV Ig :IL4, CODV Ig :IL13, CODV Ig :IL4:IL13 and CODV Ig :IL13:RefAb Fab were analysed using a OneMP mass photometer (Refeyn Ltd, Oxford, UK), previously calibrated with SB buffer to find the focal point and to estimate background noise. Subsequent to the measurements, image processing was performed as described in Sonn-Segev et al. 33 using the DiscoverMP (Refeyn Ltd) software package and graphs of intensity vs molecular weight plotted.
Characterization of antigen-antibody complexes by SPR. Antigen binding and affinity parameters for CODV Ig :IL4 and CODV Ig :IL13 were measured by Surface Plasmon Resonance (SPR) in a Biacore 8 K SPR system (GE Healthcare). A dextran CM5 sensor chip was used for the protocol, which immobilized anti-human IgG-Fc overnight via amine-coupling. Flow-cell 1 (FC1) was used as a reference while FC2 was utilized for CODV Ig and the ligands. The buffer used for the runs was STBS (50 mM Tris, 50 mM NaCl, pH 8) with the addition of Tween 20 at 0.01% to lower the surface tension and reduce non-specific binding.
For the evaluation of the dissociation constants (KD) of IL4 and IL13 binding to CODV, several runs were performed with serial dilutions of the antigens. Following the determination of the binding parameters, final SPR runs were carried out with concentrations of 1.5 µg/ml (7.6 nM) CODV Ig , 3 nM IL4 and 25 nM IL13.
Structural assessment by small angle X-ray scattering. Small Angle X-Ray Scattering (SAXS) measurements of gel-filtration purified CODV Ig :IL13:RefAb Fab were obtained using X-rays of 12.5 keV energy (λ = 0.99 Å) and a Pilatus 1 M pixel array detector (Dectris) at the ESRF BioSAXS beamline, BM29 34 . 3600 individual frames with a collection time 1 s per frame were collected. Of these, 183 frames were selected, chosen using the similarity plot in ScatterIV 35 . These were merged and subtracted from the buffer. Primary data analysis was carried out using ScatterIV and low-resolution envelopes of the solution structures obtained using DAM-MIF from ATSAS 36 online tool from EMBL Hamburg. 100 µl of freshly purified TC was used at a concentration of 1 mg/ml in the SEC-SAXS variant of the technique 37 . The column and flow rate used were identical to those used in the purification of TC (Superose 6 Increase 3.2/300 at 0.04 ml/min), while the chromatography system was a Nexera X2 HPLC (Shimadzu) and the buffer used was SB with the addition of 1 mM tris(2-carboxyethyl) phosphine (TCEP) to protect the sample from radiation damage 38 . Other parameters are listed in Table S4. nsEM Grid preparation. Of the various stains tried, 2% SST was found to be the most suitable for CODV Ig and the complexes studied here. For sample preparation, 3 µl of a solution containing antibody or antibody-antigen complexes was adsorbed to the interface between carbon and mica which, after few seconds of incubation, was slowly inserted into a well that contains the stain. A clean 400-mesh copper grid was placed on the carbon and the whole carbon-grid assembly air-dried. For good particle distribution, optimal sample concentrations were observed to be around 10 µg/ml for CODV Ig -antigen complexes and 15 µg/ml for apo-CODV Ig alone. nsEM data collection and image processing. nsEM grids were screened at the Grenoble Institut de Biologie structurale (IBS) Electron Microscope facility using a Tecnai T-12 120 keV transmission electron microscope (Thermo Fisher Scientific) fitted with a Gatan Orius SC 600 camera (AMETEK). Data collection for image analysis was performed using a Tecnai F-20 200 keV microscope equipped with a 4 K x 4 K Ceta CMOS camera (Thermo Fisher Scientific). Micrographs were recorded using the TEM Imaging & Analysis (TIA, Thermofisher Scientific) software under low-dose mode.
Estimation of CTF parameters was carried out using CTFFIND4 39 . Particles were picked fully automatically using the Laplacian or Gaussian (LoG) blob detection algorithm implemented in RELION 3.0 40 , and further 2D and 3D classifications were also performed using RELION 3.0.
Cryo-EM sample preparation and data collection. 3 µl of the samples at a 300 μg/ml concentration supplemented with 0.03 mM DDM were applied to an Au-CFlat 1.2/1.3 300 mesh grid and vitrified using a Vitrobot (ThermoFisher Scientific) using a blot time of 7 s and a blot force of 2.
Grid optimisation screening was performed at the IBS Electron Microscope facility using a 200 keV Glacios microscope equipped with both Falcon II detector (Thermo Fisher Scientific) and a Gatan K2 detector (AMETEK). All high-resolution data collections were performed on the ESRF CM01 beamline 41  www.nature.com/scientificreports/ −1.2 and −3.0 μm. The CODV Ig :IL13:RefAb Fab tilted dataset (1188 movies), was performed exploiting a 40° stage tilt and a total dose of 52 e − /Å 2 . The CODV Ig :IL4 dataset comprised 7044 movies collected over a defocus range of -1.5 to -3.5 μm and a total dose of ~ 60 e − /Å 2 and a pixel size of 0.827 Å. The CODV Ig :IL4:IL13 dataset (2919 movies) was collected using a similar defocus ranges and total dose, with a pixel size 1.053 Å.
Cryo-EM image processing and 3D reconstruction. The datasets for CODV Ig :IL13:RefAb Fab , CODV Ig :IL4 and CODV Ig :IL4:IL13 were processed until the 2D classification step within the cryoSPARC2 42 environment. Motion correction and dose weighting of movies was carried out using MotionCor2 43 while CTF estimation was carried out with the Patch CTF functionality of cryoSPARC2.
For all three data sets, initial manual particle picking was followed by an automatic protocol using the class averages from the manual step as templates (cryoSPARC2). Suitable particles identified from 2D classification were then imported into RELION 3.0 classification. For particles used in the Fab-focused refinement, 2D class averages from the full-antibody processing pipeline were first centered on the Fab density using a 110 Å mask and a high center-of-mass fraction value for recentering. The centered Fabs were then used as templates for automatic repicking of Fab densities. 2D classification in cryoSPARC2 was used to separate high-resolution classes and corresponding particles were imported further into RELION 3 for the 3D processing. In RELION 3.0, ab-initio model generation and 3D classification rounds were performed with the aim of isolating single conformations and classes that could contain densities corresponding to antigens. Finally, 3D multivariance analysis of TC was performed using cryoSPARC3 17 .
Bayesian polishing was performed within RELION 3.1. The global resolution of the final map of all the reported structures was estimated according to the gold standard Fourier shell correlation (GS-FSC) with a cutoff of 0.143 44 . Local resolution variability was estimated using ResMap 45 , with half-maps as input volumes.
EM density maps obtained were visualized with UCSF Chimera 46 and published structures of apo-CODV Fab (PDB code: 5HCG 11 ) and the knob in hole IgG-Fc (PDB code: 4NQS 47 ) were used for docking.
Model building and refinement. Models for fitting and refinement were obtained by homology modelling using SWISS-MODEL 48 , based on NCBI BlastP 49 alignment between the sequences of the proteins used for this study and the already published individual PDB structures of IL13:RefAb Fab (PDB code: 5L6Y 15 ) and apo-CODV Fab . Rigid-body fitting of the model to map was performed using Chimera and further refined by positional refinement using Real Space Refine within Phenix 50 . Map sharpening was performed either using LocScale 51 from the CCPEM 52 package or Autosharpen within Phenix 50 . Manual model building and validation were performed in Coot 53 and Molprobity 54 respectively. EM data collection parameters and refinement statistics for the TC are presented in Table S2.

Data availability
The cryo-EM reconstruction and the 3D model of CODV Fab :IL13:RefAb have been deposited in the EM Data Bank (https:// www. ebi. ac. uk/ emdb/) and PDB data bank (https:// www. rcsb. org) with the accession codes EMD-16113 and 8BLQ, respectively. All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. Request for materials will be subject to a standard material transfer agreement with Sanofi. www.nature.com/scientificreports/